Differentiation, maturation, and collection of THP-1-derived dendritic cells based on a PEG hydrogel culture platform.

PURPOSE

Dendritic cell (DC) is a spearhead responsible for immune response and surrounded by extracellular matrix in three-dimensional (3D) tissue. Nevertheless, conventional DC culture has relied on suspension or two-dimensional (2D) tissue culture plate (TCP)-based culture system. This culture condition often fails to recapitulate the physiological behavior of DC in real tissue. In this work, the effect of culture condition on DC physiology was explored with varying 3D hydrogel property (i.e., degradability, adhesion, and stiffness). In particular, DC differentiation and maturation in 3D were evaluated comparing the conventional TCP-based culture condition.

METHOD

THP-1 cells were encapsulated in poly(ethylene glycol) (PEG) hydrogel via thiol-ene photocrosslinking with non-degradable or proteolytically degradable peptide crosslinker. Hydrogel stiffness was manipulated by controlling the concentration of crosslinker. The metabolic activities and cytotoxicity of the encapsulated cells were measured by resazurin and Live/Dead assays, respectively. Cell harvesting was conducted via enzymatic degradation using α-chymotrypsin, and differentiation and maturation of the liberated DCs were evaluated by quantitative polymerase chain reaction and flow cytometry.

RESULTS

THP-1 cells well proliferated in the soft degradable hydrogel with a higher metabolic activity. However, the stiff matrix inhibited cell growth in 3D. The gene expression assay indicated that the 3D hydrogel condition was superior to 2D culture in terms of differentiation and maturation of DC. Interestingly, the stiffness of matrix was important factor in DC function. In the stiff hydrogel, the expression levels of differentiation and maturation markers were higher compared to the low stiffness hydrogel. The mature DCs caged in the hydrogel matrix were harvested after short enzymatic digestion of hydrogel and the liberated cells had over 90% viability. The flow cytometric result revealed that the proportion of CD80 + /CD86 + cells from the stiff hydrogel was relatively higher than cells either from 2D or soft hydrogel in 3D.

CONCLUSION

The collected evidence indicated that the proteolytically degradable PEG hydrogel matrix promoted DC differentiation and maturation. In addition, the matrix stiffness control could manipulate the marker expressions of differentiation and maturation. Particularly, the mature DC was successfully collected from the hydrogel matrix. These results highlighted the PEG hydrogel-based DC culture might be a useful tool for potential DC-based immunotherapies.